Divergence of upstream regulatory pathways of the transcription factor Doublesex ( Dsx ) serves as a basis for evolution of sex-determining mechanisms in animals. However, little is known about the regulation of Dsx in environmental sex determination. In the crustacean Daphnia magna , environmental sex determination is implemented by male-specific expression of the Dsx ortholog, Dsx1 . Transcriptional regulation of Dsx1 comprises at least three phases during embryogenesis: non-sex-specific initiation, male-specific up-regulation, and its maintenance. Herein, we demonstrate that the male-specific up-regulation is controlled by the bZIP transcription factor, Vrille ( Vri ), an ortholog of the circadian clock genes— Drosophila Vri and mammalian E4BP4/NFIL3 . Sequence analysis of the Dsx1... promoter/enhancer revealed a conserved element among two Daphnia species ( D . magna and D . pulex ), which contains a potential enhancer harboring a consensus Vri binding site overlapped with a consensus Dsx binding site. Besides non-sex-specific expression of Vri in late embryos, we found male-specific expression in early gastrula before the Dsx1 up-regulation phase begins. Knockdown of Vri in male embryos showed reduction of Dsx1 expression. In addition, transient overexpression of Vri in early female embryos up-regulated the expression of Dsx1 and induced male-specific trait. Targeted mutagenesis using CRISPR/Cas9 disrupted the enhancer on genome in males, which led to the reduction of Dsx1 expression. These results indicate that Vri was co-opted as a transcriptional activator of Dsx1 in environmental sex determination of D . magna . The data suggests the remarkably plastic nature of gene regulatory network in sex determination.